A crucial element in the development of effective therapeutic and prophylactic strategies for AIDS is an experimental animal model in which the course of immunodeficiency virus infection parallels the pathogenesis of the human disease. SIV infection of macaques is such a model since this virus induces an immunodeficiency syndrome in infected macaques that is remarkably similar to human AIDS. Therefore, candidate vaccines can be evaluated not only for their ability to prevent infection but also for their ability to prevent AIDS. The major vaccine effort within the laboratory has been an evaluation of the highly attenuated vaccinia virus Ankara (MVA) strain as a recombinant vector used for priming that is followed by a subunit SIV boost. Interest in this approach is based upon a pilot study of 12 macaques in which animals immunized with the MVA-SIV recombinant exhibited significant down modulation of virus replication that was associated with prolonged survival. We explored MVA as a live virus vaccine in the SIV model. Our eventual goal was to develop this approach as an AIDS vaccine for humans. Three second generation recombinant MVA viruses were generated that expressed either gag-pol alone, env alone, or a combination of gag-pol and env, with all genes being expressed from the strong early/late synthetic promoter. A cohort of 24 rhesus macaques were immunized (six per immunogen) to evaluate immunogenicity with the eventual goal of analyzing the kinetics of viremia in these animals after intravenous challenge and attempt to correlate any effects on viral load with immunogen. Four sequential immunizations resulted in moderate boosting of both gag and env-specific antibody responses. None of the animals developed neutralizing antibody specific for the challenge strain. These macaques were challenged with cell free SIVsmE660 and all of the macaques became infected. However, plasma viremia in each of the groups immunized with MVA-SIV recombinants had significantly lower viral load (p=0.03) than the group vaccinated with nonrecombinant MVA. There was no significant difference in viral load between the groups immunized with MVA-SIV recombinants. Seven vaccinees have maintained low viremia and normal CD4 numbers whereas all of the control macaques have developed CD4 depletion. These data demonstrate that vaccination with MVA-SIV recombinants results in significant protection from high viremia and AIDS presumably mediated through cell mediated immunity. Evaluation of gag-specific CTL was performed in collaboration with Dr. Norman Letvin, utilizing macaques expressing the MamuA*01 MHC Class I haplotype for which a dominant gag-specific epitope (p11 C) has been identified. Four MamuA*01+ macaques were immunized with the MVA-gag-pol recombinant virus at 0 and 13 weeks and an additional two macaques were immunized with MVA and sequential generation of a p11C-specific CTL response was measured utilizing both traditional CTL assays as well as MHC Class I/Peptide tretramer staining. Macaques immunized with MVA expressing gag-pol developed CD8+ CTL as early as 2 weeks after the first immunization (2 of 4) which was boosted in all animals to high levels after the second immunization. CTL could be demonstrated in 1 to 3% of peripheral blood lymphocytes and lymph nodes by MHC Class I/Peptide tetramer staining after the second immunization. These levels are equivalent to those observed in SIV-infected macaques. Challenge of these macaques with SIVsmE660 resulted in a rapid and substantial anamnestic CTL response. Viral load set-point was reduced in the macaques immunized with MVA-Gag-pol as compared to controls, however, the difference was not statistically significant. However, much of the spread in viremia in the immunized macaques could be explained by their response to immunization since there was an inverse correlation between the magnitude of the CTL response during vaccination and plasma viral load set-point established after SIV challenge. Finally, we evaluated the MVA strategy using a different challenge model, SHIV/89.6P infection of rhesus macaques to allow a more direct comparison with results of other vaccine strategies. Four MamuA*01+ macaques were immmunized with MVA expressing SIVmac239 gag-pol and MVA expressing HIV/89.6 env sequentially three times and CTL responses were monitored by teramer staining for the dominant Gag p11C epitope and the subdominant Env p41A epitope. All animals mounted a c11C response whereas only one animal mounted a detectable response to the subdominant env epitope. This contrasts with a uniform response to both epitopes in macaques immunized with DNA. Following challenge with pathogenic SHIV/89.6P a significant reduction in stepoint viremia and partial protection from CD4 lymphocyte depletion was observed when compared with animals immunized with MVA nonrecombinant. This degree of protection is only slightly less robust than that observed in macaques immunized with cytokine augmented DNA (Letvin lab) and DNA prime-MVA boosted macaques (Robinson and Moss lab). The ability to control SHIV89.6 infection demonstrates the usefulness and effectiveness of this vaccine strategy as a sole immunogen or as part of a prime-boost strategy. Following challenge, a substantial expansion of CTL specific for the subdominant env peptide was observed despite the fact that this epitope was not recognized pre-challenge in these animals.